Molina and Vilchez Revista Chilena de Historia Natural 2014, 87:14 http://www.revchilhistnat.com/content/87/1/14

REVIEW Open Access Cooperation and bacterial pathogenicity: an approach to social evolution C Alfonso Molina1,3* and Susana Vilchez2

Abstract

Kin selection could provide an explanation for social behavior in bacteria. The production of public goods such as extracellular molecules is metabolically costly for bacteria but could help them to exploit nutrients or invade a host. Some bacterial cells called social cheaters do not produce public goods; however, they take advantage of these extracellular molecules. In this review, the relationships between social behavior, cooperation, and evolution of bacterial pathogenicity are analyzed. This paper also examines the role of horizontal transfer of genes encoding for virulence factors and how the movement of mobile genetic elements would influence the pathogenicity and social relationships. Moreover, the link between ecological relationships and evolution in entomopathogenic bacteria, focusing on is considered. Finally, the findings obtained with B. thuringiensis are extrapolated on Bacillus pumilus 15.1, an entomopathogenic strain whose pathogenicity is not understood yet. Keywords: Cooperation; Evolution; Pathogenicity; Public goods; Sociability

Introduction (Brown 1999), adhesive polymers (Rainey and Rainey The social relationships of microorganisms are based on 2003), or siderophores (West and Buckling 2003), amongst a wide range of extracellular actions produced by indi- others. This social behavior is, from a metabolic point of vidual cells, which can affect the reproductive efficiency view, costly for the individual cells, although it is beneficial of other nearby cells. The production of molecules such for the group. However, certain cells, known as ‘cheaters’ as enzymes or chelants, which allow for the exploitation can decide not to produce these beneficial molecules for of nutrients that would not otherwise be accessible, or the group and instead take advantage of the rewards of the formation of multicellular structures, are examples social actions without paying any cost (West et al. 2006). of this kind of action (Buckling and Rainey 2002; Griffin Thus, in microorganisms, social cheaters that do not dis- et al. 2004; West et al. 2006). Extracellular products and play cooperative behavior are considered mutants (West other resources are public goods that may be used by et al. 2007; Wakano et al. 2009; Foster 2010; Smith et al. the individual that produces them or by all individuals of 2014) and can evolve with relative speed if they are favor- the group or population (West et al. 2006, 2007). For ably selected (Velicer et al. 2000; Rainey and Rainey 2003; example, bacteria produce numerous factors that are Foster et al. 2004; Griffin et al. 2004; Dugatkin et al. 2005; considered to be public goods and are released into the Harrison and Buckling 2005). environment, such as quorum-sensing molecules (Daniels Evolution experiments conducted on a wide range of et al. 2003; Diggle et al. 2007a; Williams et al. 2007), mem- microorganisms have demonstrated that this kind of brane vesicles (Schooling and Beveridge 2006), microbial cooperative behavior is most prevalent when there is a repellents (Burgess et al. 2003), host manipulation factors high degree of genetic closeness between the cells (West et al. 2006; Mitri et al. 2011). For example, the opportunis- tic pathogen Pseudomonas aeruginosa (Schroeter, 1872) * Correspondence: [email protected] 1Facultad de Ciencias Ambientales, Universidad Internacional SEK, Calle produces extracellular iron-chelating molecules, known as Alberto Einstein y 5ta. Transversal, Campus Miguel de Cervantes, Carcelén, siderophores (West and Buckling 2003). These molecules Quito 170120, Ecuador are iron-scavenging agents that are released into the envir- 3Centro Internacional de Zoonosis, Facultad de Medicina Veterinaria y Zootecnia, Universidad Central del Ecuador (UC), PO Box.17-03-100, Quito, onment in response to a lack of this element (Ratledge Ecuador and Dover 2000). The siderophores high affinity for iron Full list of author information is available at the end of the article

© 2014 Molina and Vilchez; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Molina and Vilchez Revista Chilena de Historia Natural 2014, 87:14 Page 2 of 9 http://www.revchilhistnat.com/content/87/1/14

enables them to capture the metal from compounds such cooperate to form eukaryotic cells, cells cooperate to as ferrous hydroxide and host organism proteins such as form multicellular organisms, pathogenic bacteria cooper- transferrin or ferritin (Neilands 1995; Drechsel and Jung ate to overcome the defenses of their hosts, animals feed 1998). In addition, the relationship between siderophore cooperatively, and humans and some species of insect co- production and bacterial growth rate suggests that the operate to construct societies (West et al. 2006, 2007). production of siderophores contributes to bacterial viru- With regard to the cooperation of pathogenic bacteria, lence (West and Buckling 2003). Some cells have evolved reproduction within the host can be a cooperative activity as social cheaters since they produce small quantities of that involves the secretion of virulent factors whose bene- siderophores and yet they benefit from the action of side- fits can be shared by all of the cells, whether they secrete rophores produced by other cells (Ross-Gillespie et al. them or not (Smith 2001). In this way, cooperation and 2007; Mitri et al. 2011). Using this strategy, the cheats success in overcoming the host's defenses will depend on increase their frequency in comparison with the wild the nature of the (public good) as well as genotype (that does produce the molecule) (Mitri et al. the proportion of social cheaters present in a population. 2011; Smith et al. 2014). However, in order for sidero- For example, this could explain why the enterotoxigenic phore production to remain high, populations must be Escherichia coli (Migula, 1895) (producer of LT and ST exposed to a strong genetic bottleneck, which ensures that ) requires a greater population density to cause an the genetic relationship between the cells that produces infection when compared with the enterohemorrhagic E. the chelating agent and the one that is not is high. There coli, a producer of Shiga (STX) (Smith and Halls are numerous examples of the role of kin selection in 1968; O'brien et al. 1984), which requires a smaller num- explaining the cooperation between microorganisms, such ber of cells. Enterohemorrhagic E. coli populations would as the example of Bacillus subtilis (Ehrenberg, 1835), therefore include a lower proportion of cheater cells. which forms biofilms in which closely related cells cooper- Although there is no doubt about cooperative behavior, ate by becoming differentiated and sharing out functions there is controversy on how this kind of behavior might (Foster 2010). This same kind of social behavior has been be interpreted, via kin selection or via group selection reported in various other species (Wakano et al. 2009). (West et al. 2007; Wilson 2008). As mentioned above, This sacrificial phenomenon does not occur in multi- there are various pieces of evidence that support the idea cellular organisms since the cells do not differ genetic- that this kind of behavior in bacteria could be explained ally from one another. No cell has an advantage over by the kin selection theory. For example, in bacterial spe- another, and if any cell should vary, it would be excluded cies that form biofilms, cells that are genetically related from the cell line. For this reason, the deception of indi- are found close to one another (Griffin et al. 2004; Diggle vidual cells is restricted in the majority of multicellular et al. 2007a,b). Nevertheless, the fact that all of the cells organisms (Kessin 2000). However, there is another originate from a single mother cell in the majority of bio- route to multicellularity that is adopted by organisms films has been presented as an argument in favor of group such as Dictyostelium or Myxococus (a group of social selection (West et al. 2006; Boyle et al. 2013; Drescher bacteria). When nutrients become limited and cell dens- et al. 2013). ity is high, Myxococcus xanthus (Beebe, 1941) activates Biofilms are communities of microbial cells, commonly various genes that break up the aggregation of local composed of various lineages or species, which grow on groups that exchange intercellular signals, allowing the surfaces surrounded by polymers (Kolenbrander 2000; formation of spore-producing fruiting bodies (Kadam and Hall-Stoodley et al. 2004). In this kind of community, the Velicer 2006). These fruiting bodies contain approximately cellular groups have limited space and influence each 50,000 cells, but only a fraction of the cells have differenti- other depending on the distance between them. These ated latent myxospores (Curtis et al. 2007) which appar- spatial relationships are of the utmost importance for the ently sacrifice themselves for the common good (Fiegna compression of cellular community cooperation evolution and Velicer 2006; Fiegna et al. 2006; Kadam and Velicer (Durrett and Levin 1994; Drescher et al. 2013). When dif- 2006). M. xanthus cells also secrete toxins that can kill ferent cellular lineages are separated in space, cooperative other cells within the fruiting body, or the species that phenotypes are more likely to benefit other cells belonging feed on them; therefore, it is considered an example of to their phenotype (Mitteldorf and Wilson 2000; Griffin group predation (Foster 2010). In addition, there are mu- and West 2002; Axelrod et al. 2004). However, when the tant social cheaters that make up part of the local group different cellular lineages are intermingled, cells that ex- and contribute less to the production of the non-spore- ploit the resources of others are able to prosper more effi- producing parts of the fruiting body (Fiegna et al. 2006; ciently (Griffin et al. 2004; Diggle et al. 2007a,b; Sandoz West et al. 2007). et al. 2007; Chuang et al. 2009). If the cells of a genotype Cooperation can be found at all levels of biological are mixed with other genotypes, it is more probable that organization: genes cooperate in genomes, organelles competitive characteristics that cause potential harm to Molina and Vilchez Revista Chilena de Historia Natural 2014, 87:14 Page 3 of 9 http://www.revchilhistnat.com/content/87/1/14

neighboring cells will evolve than when the cells are sur- that bottlenecks can also be indicative of the fact that rounded by clones (Mitri et al. 2011). Therefore, it has strong ecological competition can eliminate cells with co- been suggested that the spatial structure of biofilms is a operative behavior before they have had the chance to es- critical factor in microbial interactions (Boyle et al. 2013). tablish themselves. For example, it has been demonstrated Intra-species cooperation could be favored by spatial seg- that P. aeruginosa lineages that secrete siderophores that regation that keeps secreting cells away from those that capture iron are vulnerable to competition from lineages do not secrete, while inter-species cooperation is favored that do not secrete (but that use the siderophores without by the presence of various species (Sandoz et al. 2007; producing them) when Staphylococcus aureus (Rosenbach, Nadell et al. 2009). Cooperative phenotypes are subject to 1884) is added (Harrison et al. 2008). Population bottle- exploitation by non-cooperative cell lineages since they necks fulfill a fundamental role in the maintenance of so- could be influencing the interaction between cooperative cial characteristics in microorganisms. Some ecological and non-cooperative cells (Nadell et al. 2009, 2010). It is parameters such as colonization or disturbance may favor difficult for cooperative cells to be successful in competi- cooperation, causing population bottlenecks that increase tion against non-cooperative cells, which exploit public genetic kinship. Moreover, the size of the population goods without paying the costs. However, if the coopera- bottleneck fulfills a fundamental role in the success of co- tive cells are spatially segregated and prefer to interact operation. In this way, kinship increases as the size of the amongst themselves, they can prevail. The spatial distribu- bottleneck decreases, favoring the evolution of cooper- tion of genetic lineages is related to physical and biological ation. Brockhurst (2007) used Pseudomomas fluorescens parameters, such as the availability and capacity for nutri- (Migula, 1985) SBW25 to experimentally prove that the ent diffusion, cellular metabolic efficiency, cell growth quantity of social cheaters increases as the size of the rate, or biomass density. Drastic changes in these parame- bottleneck increases, which suggests that the reduction in ters can change the spatial layout of microbial populations kinship caused by large genetic bottlenecks works against within a community and determine if cells with coopera- cooperation. tive phenotypes could compete locally or globally with Other experiments have underlined the importance of social cheaters. The result would be the segregation ecological competition in favoring cooperation between of cellular lines that favor the evolution of cooperative species. The model developed by Rankin et al. (2007) de- phenotypes (Nadell et al. 2009). monstrated that intra-species competition can increase or There is also abundant evidence that demonstrates the decrease the ability to compete with other species. It has fact that the presence of different genotypes in biofilms also been proposed that interactions with other species limits cooperation across a wide range of bacterial charac- can promote the evolution of secreting genotypes (Mitri teristics (Greig and Travisano 2004; Gore et al. 2009), such et al. 2011; Drescher et al. 2013). Social isolation allows as the secretion of enzymes (Griffin et al. 2004); iron cap- the secreting cells to form patches in which they preferen- ture (Diggle et al. 2007a), the secretion of quorum-sensing tially help their own genotype; therefore, the spatial struc- molecules and the formation of fruiting bodies (Foster tureofabiofilmmaypromotetheevolutionofcooperation et al. 2002; Buttery et al. 2009). Accordingly, it has been (Nowak and May 1992; Xavier et al. 2011). The importance suggested that interactions between species can have a of the effects of social isolation in natural communities has profound influence on intra-species cooperative behavior not yet been clarified. However, it has been suggested that in spatially structured microbial groups like biofilms. It it could be more significant under conditions where nutri- has been demonstrated that ecological competition with ents are abundant in which various species can coexist. An other species preferentially prejudices secreting cells more interesting case study is that of the human microbiome, than non-secreting cells or social cheaters. This may be particularly that of the intestine, where the cells can form due to the fact that investing in secreting extracellular dense biofilms containing various species (Macfarlane and molecules can delay the growth of cellular lineages at Dillon 2007). critical stages. This initial investment leaves secreting cells As mentioned above, the secretion of public goods in vulnerable to competition with other lineages, particularly biofilms is affected by the availability of nutrients. When in nutrient-poor conditions where resources are limiting competition for nutrients is strong, the addition of new and the majority of lineages are eliminated through a species can inhibit cooperation by eradicating secreting strong genetic bottleneck. The potential for bottlenecks in lineages before they can establish themselves. When nu- growing microbial groups has been well documented trients are abundant and various species are found in (Gage 2002; Hallatschek et al. 2007; Boyle et al. 2013). the same habitat, the secreting lineages of any species Bottlenecks have been interpreted as favorable for the are surrounded by other species. This ‘social isolation’ evolution of cooperation because they promote genetic protects those cells that produce public goods from identity in emerging clonal groups (Brockhurst 2007; competition by those from the same species that do not Nadell et al. 2010). However, Mitri et al. (2011) suggest produce public goods, and this can improve cooperation Molina and Vilchez Revista Chilena de Historia Natural 2014, 87:14 Page 4 of 9 http://www.revchilhistnat.com/content/87/1/14

between species (Hol et al. 2013). In addition, limitations understood why these genes are so mobile (Griffin et al. in the interactions between species have been observed 2004; West et al. 2006; Nogueira et al. 2009). since it is difficult to find conditions that encourage co- While plasmids benefit from the horizontal gene trans- operation between cells of the same species and of fer as selfish DNA, this mobility implies a range of costs other species (Mitri et al. 2011). On the other hand, on host bacteria, especially in terms of the resources it has been suggested that species with different metabolic invested in conjugation (Nogueira et al. 2009). On the needs show a greater tendency toward mutualistic behav- other hand, bacteriophages are major contributors to the ior (Little et al. 2008). In general, cooperation in microbial process of horizontal gene transfer given their environ- communities will be favored when there is a high level of mental ubiquity, their great abundance in nature, and competition between communities (Mitri et al. 2011). the functional effects that they cause in hosts (Hanage In conclusion, the appearance of biofilm organization et al. 2005). In this way, phages make a significant con- could have arisen without active coordination. This tribution to the diversity and evolution of bacteria since, implies that certain properties like phenotypic differ- in addition to acting as vectors for gene transfer, they entiation, species stratification, and the formation of promote a high coevolution rate and a high level of channels do not necessarily require cells to communicate specialization between bacterial genotypes in natural amongst themselves using specialized signal molecules. microbial communities (Siefert 2009). Lysogenic phages Also, although local cooperation between bacteria occurs integrate their genome within the host genome until the frequently, the evolution of cooperation between cells be- prophage is excised from the host genome and cell lysis longing to a biofilm may be unlikely. Strong conflicts may results. During this process, various segments of the host arise between species and lineages in a biofilm, and spon- DNA can be introduced into the viral genome, and when taneous mutations may cause conflicts even if the com- lysis of the cell occurs, the host DNA is dispersed in the munities were initiated by cells that were genetically phages (Siefert 2009). In this way, during lysogenic con- identical (Nadell et al. 2009; Hol et al. 2013). version, prophages can provide benefits to their hosts On the other hand, the appearance of cooperation via while they remain latent through the addition of new kin selection has also been described in more complex functions in the bacterial genome. For example, it is organisms such as (Griffin et al. 2004). With- through this process that the harmless Vibrio cholerae out a doubt, the best way to clarify what kind of selection (Pacini, 1854) lineage is transformed into the highly is affecting the evolution of cooperative behavior is by virulent lineage that causes cholera (Hanage et al. 2006). means of experimentation. The evidence provided by Additionally, the insertion sequences (which can be auto- experimental studies of evolution (Griffin et al. 2004; nomous or part of composite transposons) are efficient at Kummerli et al. 2009) has been a fundamental pillar in the moving themselves between bacterial genomes as they understanding of microorganism sociability. reshuffle and shape them (Chandler and Mahillon 2002; Siguier et al. 2006). It has been demonstrated that the massive expansion of insertion sequences has a positive Review correlation with the appearance of some pathogenic bac- Horizontal gene transfer and the evolution of bacterial terial species (Siefert 2009). pathogenicity Numerous theories could explain why bacteria invest Bacteria transfer their genes vertically, from a mother cell in gene mobility but none has been experimentally to a daughter cell, similarly to what happens in more com- tested, which means that the evolutionary bases of the plex organisms, although they can also interchange their fundamental genome organization of bacteria remain un- genes horizontally. Genes move quickly between genomes clear. The horizontal gene transfer is particularly import- via mobile genetic elements such as plasmids, transpo- ant in the evolution of infectious diseases, in the spread of sons, bacteriophages, and self-splicing molecular parasites virulence genes, and in antibiotic resistance (Holden et al. (Nogueira et al. 2009; Siefert 2009). A vertically transmit- 2004; Frost et al. 2005; Gogarten and Townsend 2005; ted genome encodes for fundamental cellular processes, Murphy and Boyd 2008). Key virulence genes, essential while a horizontally transmitted genome encodes for genes for infecting host organisms, are carried on mobile genetic that allow for the exploitation of specialized niches or elements in various clinically significant species such as S. genes that provide resistance to toxic molecules (Hacker aureus, Bacillus cereus (Frankland and Frankland, 1887), and Carniel 2001). Many of the genes responsible for E. coli, and the agents that cause anthrax, cholera, and pathogenic bacterial virulence are found in mobile genetic diphtheria (Smith 2001). elements that can be transmitted horizontally between Virulence, secretion, and horizontal mobility are closely different bacterial lineages. The horizontal transfer of viru- related in bacterial genomes. Virulence factors are com- lence factor genes has played a fundamental role in the monly secreted with the aim of reaching specialized tissue evolution of pathogenic bacteria. However, it is poorly within the host (Smith 2001). There is evidence that reveals Molina and Vilchez Revista Chilena de Historia Natural 2014, 87:14 Page 5 of 9 http://www.revchilhistnat.com/content/87/1/14

that the E. coli genes responsible for secreted products biological control agent. B. thuringiensis is a strict aerobic, tend to be more mobile than genes coding for intracellular Gram-positive, flagellate, ubiquitous, sporulating bacteria products (Nogueira et al. 2009). Secreted products are used that is morphologically and genetically related to B. cereus as public goods by neighboring bacteria, independently of and B. anthracis (Cohn, 1872) and is widely used as a bio- whether the neighbor secrets products for itself. In co- logical insecticide for insect control (Schnepf et al. 1998; operative secretion, the individual bacteria that invest in Crickmore et al. 2013). B. thuringiensis produces protein secretion pay a cost in terms of growth, although the crystals with insecticidal properties (Helgason et al. 2000). group benefits from the presence of virulence factors, for The proteins of which they are composed are called instance allowing an infection to become established in a δ-endotoxins and there are two main types: Cry toxins host organism (West et al. 2006; Raymond et al. 2010). and Cyt toxins. δ-endotoxins are pore-forming proteins Virulence factor genes would have evolved to increase specifically active toward membranes from insects the pathogenicity of bacteria, causing positive effects (Lepidoptera, Coleoptera, and Diptera), acari, nema- such as an increase in their transmission rate, which todes, flat worms, and protozoa (Schnepf et al. 1998; would help them to colonize new hosts, obtain new re- Lightwood et al. 2000; Li et al. 2001). sources, evade the host defenses or disperse themselves to The mechanism of action of Cry toxins is a complex colonize new individuals (Ochman et al. 2000; Nogueira process that develops in various stages; however, the et al. 2009). However, extracellular virulence factors are symptoms caused in the insect subjected to ingestion of potentially available to members of the pathogen popula- the toxins can be summed up in the following steps: (1) tion that do not produce the effector molecule. Thus, a ingestion ceases, (2) gut paralysis, (3) excretion of resi- social cheater cell could avoid the metabolic cost of dues (vomiting and diarrhea), (4) total paralysis, and (5) producing the virulence factor, but it would increase its death. A larva intoxicated with B. thuringiensis shows a frequency during an infection due to the action of characteristic black color due to tissue necrosis (Bravo molecules produced by other cells in the population et al. 1992). (Ross-Gillespie et al. 2007, 2009). Cry insecticide toxins are the main virulence factors of If pathogens compete for resources within a host, the B. thuringiensis and can be considered a public good. increase in cheats will reduce the transmission of the The sociability of these effector molecules has been genotype that produces the virulence factor (Buckling studied under almost natural conditions using the larva and Rainey 2002; Mitri et al. 2011; Smith et al. 2014). of the diamondback moth, Plutella xyllostella (Linnaeus, Nevertheless, it would be possible to reestablish the 1758), as a host (Raymond et al. 2012). As is the case infectiveness by reducing the density of the cheater cells. with other bacteria that possess social behavior, certain The pathogenicity could also be restored by reintroducing B. thuringiensis genotypes do not produce Cry toxins. functional versions of the genes coding for virulence fac- However, they are able to benefit from the production of tors in the cheats. As a result, it has been proposed that the insecticidal proteins synthesized by other individuals virulence factors are maintained in horizontally transmis- in the population when infecting the host. Thus, non- sible mobile genetic elements so that they can be reintro- toxin-producing cells can take advantage of the forma- duced and prevent a high frequency of cheats (Smith tion of pores in the epithelial cells of the insect gut to 2001; Wakano et al. 2009). Accordingly, the existence of infect it. This would demonstrate that the reproductive the tragedy of the commons in antibiotic-resistant plas- efficiency of microorganisms can depend on the cooper- mids was recently stated (Smith 2011). Mobile genetic ation between cells, both in controlled conditions and in elements would be superinfecting bacteria that had nature (Griffin et al. 2004; Raymond et al. 2012). Inter- already been previously infected, increasing the fitness of estingly, the high densities reached by social cheaters the plasmid. However, they would become victims of their could explain why B. thuringiensis does not cause epi- own success since they reduce the density of their bacterial demics on nature (Raymond et al. 2012). This connection hosts (Smith 2011). between cooperative virulence and epidemiology could also be relevant for species of bacteria that do not produce The sociability of entomopathogenic bacteria and its toxins, including some human pathogens. ecology The entomopathogenicity of Bacillus pumilus (Meyer Relatively few species of bacteria have been described as and Gottheil, 1901) could also be explained by the social insect pathogens, but they have received a lot of attention evolution of virulence factors. B. pumilus is a ubiquitous as a result of their potential for pest control in agriculture bacteria with a wide range of significant activities from and for the control of vector-transmitted diseases (van a biotechnological point of view. Some strains of B. Emden and Service 2004). Amongst these, most interest pumilus have fungicidal properties and have been used has been concentrated on Bacillus thuringiensis (Berliner, as biological control agents against phytopathogenic 1915), due to its specificity and its applicability as a fungi (Bottone and Peluso 2000; Lehman et al. 2001; Molina and Vilchez Revista Chilena de Historia Natural 2014, 87:14 Page 6 of 9 http://www.revchilhistnat.com/content/87/1/14

De-Bashan et al. 2010), while others have shown power- the aerial parts of the , where susceptible hosts are ful antibacterial activity (Aunpad and Na-Bangchang present, is a key feature of its ecology. 2007). In addition, B. pumilus has been reported as Similarly to B. thuringiensis, B. pumilus is an ubiquitous entomopathogenic bacteria. The first study that demon- bacteria whose typical habitat is the soil. The B. pumilus strated the entomopathogenicity of B. pumilus is de- 15.1 strain was isolated from a partially decomposed reed tailed in a patent that describes a strain active against plant (Molina et al. 2010), suggesting that can also the corn rootworm (e.g., Diabrotica undecimpunctata form part of the natural habitat of this bacterium. Besides, (Mannerheim, 1843), Diabotrica longicornis (Say, 1824)), as it has been mentioned above, other B. pumilus strains the armyworm (Spodoptera exigua (Hubner, 1808)), and have been reported playing many other roles: first, as a some species of nematodes (Heins et al. 1999). The en- growth-promoting rhizobacteria; second, providing pro- tomopathogenic activity of B. pumilus was confirmed in tection to the plants as a result of its antifungal properties a study that described the isolation of the strain 15.1, a against phytopathogenic fungi; and finally playing a role in highly toxic strain against Mediterranean fruit fly larvae, the plant defense reactions (Benhamou et al. 1996). Ceratitis capitata (Wiedemann, 1824) (Molina et al. It is a fact that B. pumilus interacts with plants and 2010). Since B. pumilus has never been considered as a there is evidence to suggest that this ecological relation- classic entomopathogenic bacteria, the origin and the ship is beneficial to both organisms. Smith and Couche possible evolutionary route of the insecticidal activity of (1991) proposed an explanation for this type of interaction the B. pumilus 15.1 strain is a fairly interesting topic that between a plant and an entomopathogenic bacteria. This would help to explain the process of pathogenicity. The study proposed that B. thuringiensis populations found in specific case of B. thuringiensis, whose ecology is widely the phylloplanes of plants could inhibit the feeding of known, could represent the best analogous example insect larvae. This situation could be a symbiotic relation- for the discussion of this topic. Therefore, we will ship in which B. thuringiensis also benefits from being a concentrate on B. thuringiensis strains active against phylloplane epiphyte. In such a way, the plant could pro- leaf-eating insects (since it is the best case study), with the vide nutrients from leaf exudates and associated micro- aim of explaining the possible origin of B. pumilus 15.1 flora and also provide a niche free from competition with pathogenicity. other soil-borne spore-forming bacteria. On the phyllo- The role of B. thuringiensis in the environment remains plane, B. thuringiensis is accessible to leaf-feeding insect unclear due to, among other reasons, a lack of manipula- larvae. If the larvae ingest sufficient toxin spore and crys- tive field experiments (Travers et al. 1987; Raymond et al. tals to inhibit their feeding, defoliation would be reduced, 2010). One of the many hypotheses that attempt to ex- and as a result, the plant would be protected. This pro- plain the role of B. thuringiensis suggests that the bacteria posed hypothesis for plant-entomopathogenic bacteria has evolved in order to provide symbiotic protection to association could also explain the relationship between plants since it forms part of the normal phylloplane B. pumilus and plants. Furthermore, this hypothesis microbiota (Smith and Couche 1991; Elliot et al. 2000). does not exclude other concepts about the ecological Another hypothesis proposes that B. thuringiensis is a nat- role of B. pumilus, for instance, that it could act as an an- ural soil inhabitant with incidental insecticidal activity tifungal or an antibacterial agent in the soil microcosm (Martin and Travers 1989). This hypothesis states that (Elliot et al. 2000). except from the fact that B. thuringiensis has previously Elliot et al. (2000) extended the plant hypothesis been found in association with insects, there is no reason (originally described as predators and parasitoids) to to believe that this association is vital. The ubiquity of B. entomopathogens and essentially agrees with Smith and thuringiensis in soil is consistent with this idea and further Couche (1991). In order for a plant to employ an entomo- supports the hypothesis of Dulmage and Aizawa (1982), pathogen as a bodyguard, this relationship must represent which proposes that the soil is the normal environment of a good return on investment (the benefits of the relation- B. thuringiensis. However, this bacteria is not usually toxic ship must outweigh the costs) (Price et al. 1980, Elliot against insect larvae that live in the soil, but it is against et al. 2000). The ‘bodyguard’ hypothesis in which a bac- insects with aerial or water-borne larvae. teria acts mutualistically to a plant could explain why B. On the other hand, Jensen et al. (2003) suggested that thuringiensis is maintained in the phylloplane and on the B. thuringiensis could be part of the commensal gut ground. The plant benefits from the entomopathogenic microbiota of various insect species without causing activity of the bacteria before its population reaches high obvious disease or death. However, Raymond et al. (2010) densities (Elliot et al. 2000; Buckling and Rainey 2002). demonstrated that B. thuringiensis behaves as a specialized Based on the aforementioned case of B. thuringiensis, insect pathogen in the field. This study suggests that the on the fact that B. pumilus is a ubiquitous bacteria with normal habitat of B. thuringiensis is the soil, and that the ground as its typical habitat, and on its ecological re- movement from the soil (which acts as a reservoir) toward lationship with plants, it is suggested that the bodyguard Molina and Vilchez Revista Chilena de Historia Natural 2014, 87:14 Page 7 of 9 http://www.revchilhistnat.com/content/87/1/14

hypothesis could also explain the entomopathogenicity de Cervantes – Carcelén, Quito, Ecuador. SV is affiliated to Departamento of B. pumilus 15.1. C. capitata larvae are not leaf-feeding, de Bioquímica y Biología Molecular I, Facultad de Ciencias, Universidad de Granada, Campus Universitario Fuentenueva, 18071 Granada, España. but they are considered as phytophages. For this reason, and because of the relationship that B. pumilus maintains with plants, the application of the bodyguard theory could Acknowledgements be feasible. This hypothesis may represent the best explan- The authors thank the three anonymous reviewers for their valuable comments and suggestions to improve the quality of the manuscript. ation for B. pumilus insecticidal activity. On the other hand, in spite of the fact that B. pumilus is a different Author details 1 strain, it has been reported as a pathogen for S. exigua Facultad de Ciencias Ambientales, Universidad Internacional SEK, Calle Alberto Einstein y 5ta. Transversal, Campus Miguel de Cervantes, Carcelén, (Heins et al. 1999), a species with defoliating larvae. Quito 170120, Ecuador. 2Departamento de Bioquímica y Biología Molecular I, In addition, the presence of crystals has been described Facultad de Ciencias, Universidad de Granada, Campus Universitario 3 in sporulating cultures of B. pumilus 15.1 that are simi- Fuentenueva, Granada 18071, Spain. Centro Internacional de Zoonosis, Facultad de Medicina Veterinaria y Zootecnia, Universidad Central del lar to the Cry proteins of B. thuringiensis (Molina et al. Ecuador (UC), PO Box.17-03-100, Quito, Ecuador. 2009). The role of these structures has not yet been elu- cidated. However, it could be suggested that they are in- Received: 13 January 2014 Accepted: 23 June 2014 volved in the entomopathogenicity of B. pumilus 15.1 due to their structural similarity with the insecticidal toxins of B. thuringiensis (Molina et al. 2009). 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